1. Diagnosis and treatment of disorders of amino acid metabolism in autism
Y.B. Grechanina
«IF A DRUG FITS TO EVERYBODY, IT MEANS IT DOESN’T FIT TO ANYBODY».

2. When AUTISM and autistic features of behavior have the metabolic base – this, on the one hand, worsens course severity, on the other hand – gives the concrete direction of the treatment .

3. Since a human began to intervene into actions of a natural selection, it has to change levels of its influence turning one stage of ontogenesis to another.
Attempts of saving interruptive in early terms of a pregnancy with the help of medicines lead to pretended victory – the frequency of birth of children with the inborn and inherent pathology by our data increases 4 times. (E.Y. Grechanina, 2012)

4. Signs of metabolic disorders we can observe already prenatally and in the newborn period

5. Urine color Color Compound Disorder, the source of disorders Blue
Indican
blue diaper syndrome
Brown-blue
Homogentisic acid
Alcoptunuria
Brown
Methemoglobin
Myoglobinuria
Brown-red
Hemoglobin/ methemoglobin
Hemoglobinuria
Red
Erythrocytes
Hematuria
Porphyrines
Porphyry
Pyrosolons
Medicines
Phenolphthalein
Chemical substances
Light red
Urats
Physiological, hyperuricosuria
Beet
Caused by feeding
Yellow
Riboflavin
Vitamins

6. Urine odor Musty, mouse Phenylacetic Classical PKU
Maple syrup, burnt sugar
2- Oxoisocanronic acid 2-Oxo-З-methylvaleric acid
«Maple syrup» disease (МSUD)
Sweaty legs
Isolaleric acid
Isolaleric acidemia.
3-oxi-З-methylglutaric aciduria, multiple defects of acyl-CoA-dehydrogeneration (MAD)
Cat urine
З-Oxiisovaleric acid
3-Methylcrotonylglicinuria, multiple deficiency of carboxylase
Cabbage
2-Oxibutyric acid
Malabsorption of methionine, Tyrosinemia 1
Spoiled oil
2-Oxo-4-methylbutyric acid
Tyrosenemia 1
Acid
Methylmalonic acid
Methylmalonic aciduria
Sulfur
Hydrogen sulfide
Cystinuria
Fish
Trimethylamine
Trimethylaminuria

7. Pathogenesis of metabolic diseases: can be manifested symptomatically
Mutant allele
Pathologic primary product
(excessive,insufficient,abnormal,is absent)
Disorder of the combination of biochemical processes
Pathology inside a cell
Pathology of organs
Pathology of the body

8. Obstetrical anamnesis in metabolic diseases
- Spontaneous abortion or deadborn in the anamnesis should be considered as elimination of an unviable child.
Male gender of such fetus can say about X-linked form of metabolic diseases;
-The presence of pathologic changes in a pregnant, such as continued toxicosis or an acute fatty dystrophy of the liver, can be the result of the disorder of fatty acid oxidation in a fetus.

9. Mechanisms of the onset of metabolic crisis in IMD (по Johannes Zschocke, Georg F. Hoffmann, 1999)

10. Mechanisms of the onset of metabolic crisis in IMD (по Johannes Zschocke, Georg F. Hoffmann, 1999)

11. When it is necessary to suspect metabolism disorder?
Lethargy
Refuse from food
weight loose
breath disturbance
hypothermia
hypotonia
unusual motions
hepatomegaly
convulsions
polyorgan changes
coma

12. Stages of laboratory study
Preanalytical stage
Diagnostic significance and specificity of values
Postanalytic stage
Analytic stage

13. Preanalytical stage: Somatic and genetic study
syndromologic, clinical and genealogical analysis
Preparation of a patient for the study
Sampling of the biochemical material
Preservation and transport of samples

14. Organic acids – low molecular compounds, which are products metabolism of amino acids, hydrogen, lipids, biogenic amines.
Organic acidurias (acidemias) – a group of inherent diseases, which is characterized by the disorder of intermediate metabolism with the accumulation of carboxyl acids. Toxic compounds disturb intercellular metabolic pathways, including glucose catabolism (glycolysis), glucose synthesis (gluconeogenesis), metabolism of amino acids and pyrimidines and also fats .

15. Types of organic acids (ОА)
ОА, caused by the deficiency of enzymes participating in transformation of amino acids (leucine, isoleucine, valine, lysine, tyrosine, aminobyturic acid).
ОА, caused by the disorder of bioenergy processes (Creb’s cycle), cellular breath, oxidative phosphorylation in mitochondria of cells.
ОА, caused by the disorder of transport or mitochondrial oxidation of fatty acids.

16. 1 group- clinical manifestations:
(or at the early age)
 acute onset
 convulsions
 apnoe, dyspnea
 increased irritation (or inhibition) of CNS
 muscle hypotonia
 anorexia
 vomiting
 sometimes extrapyramidal disorders

17. 2 group – clinical manifestations:
Manifestation is preferentially at the children age;
Development delay;
Abrupt muscle weakness;
Respiratory disorders;
Cardiomyopathy, rhythm disorders;
Nervousness or sleepiness;
Convulsions, ataxia;
Nistagmus, atrophy of visual nerves;
Acidosis, accumulation of lactate, pyruvate .

18. 3 group – clinical manifestation:
Different time of manifestation;
 vomiting;
 muscle weakness;
 hypotonia;
 episodes of muscle pains and myoglobinuria;
 Reye’s syndrome;
 hepatomegaly, fatty infiltration of the liver;
 hypoglycemia with hypoketonemia

28. There are the following disorders of AA metabolism
Breakdown of protein lead to the formation of a great amount of nitrogen – a substance, which is highly toxic for CNS. Nitrogen is usually converted in urea and released with urine.
*Defects of enzymes of urea cycle and other disorders of detoxification of ammonia are manifested clinically in the form of encephalopathy and hyperammonemia
*Study of metabolism should include analysis of amino acids of blood and urine in determination of orotic acid in urine.

29. Disorder of transport of amino acids
Defects of intestine and/or renal transport of AA can be:
asymptomatic
Manifested clinically as a deficiency of essential amino acids or as a result of the disorder of AA transport (e.g. tryptophan in Hartnup disease)
Followed by the increase of uric concentration of unsolved AA (e.g. cystine in cystinuria)

30. In the result of accumulation of toxic metabolites in inborn errors of AA metabolism
Pathologic changes of different organs and systems are developed;
The risk of the development of encephalopathy increases;
Stable neurological disorders appear

31. Clinical features of some aminoacidopathies
Combination of mental retardation (MR) with convulsions (non ketotic hyperglycinemia, PKU, disorder of metabolism of AA of urea cycle, hyperlysinemia);
Combination of MR with pathology of vision (homocystinuria);
Combination of MR with skin affection (PKU, inherent xanturenuria, histidinemia);
Combination of the affection of the liver and CNS (argininemia);
Hearing disorder (hyperprolinemia).

32. Alanine/lysine ratio show energy metabolism disorder (is followed by the increase of pyruvate)
The increase of glycine level (+alanine) show hyperammonemia

33. Methods, which are used for diagnosis of disorders of AA metabolism
 Urinolysis – the qualitative and quantitative reactions. Material for study – morning urine
 Thin-layer chromatography. Material – blood, daily urine.
 Classical biochemical values and enzymes (glucose, Са, Р, LDH, CK and other)
 Quantitative analysis of AA by HELC method, Waters.
 Mass screening – newborn programs: diagnosis of PKU. Material – dry blood spots
 Perspective studies – the qualitative analysis of organic and fatty acids using tandem mass-spectrometry

34. The content of amino acids in biological liquids depends on metabolic condition:
If sampling is performed after feeding, the content of essential amino acids increases (LYS, PHE, TYR, VAL, LEU, ILE, GLN, CIT);
Long-term fasting with ketosis – the increase of amino acids with branching chain (VAL, ILE, LEU)
Unspecific changes:
hemolysis, late centrifugation cause:
ARG, ASP, GLU, ORN, TAU;
Long-term preservation of samples at room temperature -  GLN, ASN, CYS, HOCYS; ASP, GLU

35. Profile of amino acids
Alimentary upload
Liver disease
Use (medicines,diet and other) medium-chain triglycerides
Use of EDTA as a coagulant in sampling
Treatment with benzoate, pyropyruvic and valproic acids
Carnitine defect

36. Chromatographic profile. Prolinemia

37. Chromatographs of blood serum are within norm in PKU

38. Laboratory criteria of establishment of the diagnosis of concrete aminoacidopathy

39. The scheme of examination of the patient with suspicion of disorder of metabolism of sulfur-containing AA 
Thrombocytopenia
Hypersegments of neutrophils
Macrocytic anemia
Routine laboratory tests
 -norm
-norm
Methionine (blood) 
Methylmalonic acid (urine)
Homocysteine (urine, blood)
Special laboratory tests
Megaloblastic anemia
Behavioral disturbances
Development delay
Unique clinical signs
Children
Newborns
Symptoms/markers
System

40. The scheme of examination of the patient with suspicion of disorder of metabolism of sulfur-containing AA 
Retina degeneration
Eyes
Acute psychosis
Dementia
Speech disturbance
Myelopathy
Spasticity
Convulsions
Lethargy
Hypotonia
Mental retardation
CNS
Children
Newborns
Symptoms/markers
System

41. Characteristic deficiency of amino acids in autism
According DAN theory: taurine is decreased and its intake is recommended in a high dose in chelation
In statistical study of Moreno-Fuenmayor et al., performed in 1996 year,it was proved that 50% of children had an increased level of taurine, that was explained by compensatory character.
Statistical study of 330 analyses in KhSMGC has shown its increase in 56% cases (that corresponds to serious study data), and its decrease only in 1 case.

42. In considering other amino acids, DAN-theory is plastic in explanation (the decrease of amino acids in plasma, not connected with a diet, DAN- theory connects with two well-known effects of mercurous: inhibition of chorohydric acid development in ventricle, inhibition of various proteases and peptides, that creates problems for amino acid absorption, but self-confident in treatment: “tests can be not the best indicators, real test of therapy is more reliable”.

43. DAN-theory recommends to increase the amount of proteins (proteins in food).
Producers of BAA, more often using Bioshape and Protivity, also take part (isoleucine, methionine, valine, tryptophan, phenylalanine, lysine, valine), persuade the consumer in that there is no a single person, whom wasn’t prescribed periodical intake of amino acids.

44. Examination of children older than 10 years, performed in 1996, showed that the concentration of glutamate and aspartate were appeared to be enough high, and glutamine and asparagine – low, the half of children had the increase of taurine.
There is a hypothesis that abnormality of glutamate levels can be caused by the presence high amounts of this amino acid in food, can have endogenic pattern (the result of disorder of metabolism of glutamate, receptor blockage and carrier function change).The increase of taurine concentrations, most probably, has compensatory pattern.

45. The conclusion was made: children with autism are born in families with disorder of the regulation of amino acid metabolism, tat indicates the biochemical basis of this disease.

46. In I.S. Boksha’s opinion, 2005, such changes of amino acids correspond to glutamate neuromediate system and disorder (or change of synthesis speed) of the structure of neuromediate system components (receptors and carriers), including glutamate and cholinergic, serotonergic, dopaminergic, and also neuromediator metabolism play the key role in autism development.

47. The decrease of essential amino acids is confirmed by studies (G
The decrease of essential amino acids is confirmed by studies (G. Novarino et al, 2012), this is explained by the mutation in BCKDK gene, which inactivates BCKD- kinase.

48. When DAN-recommendations help!
If L-glutamine is in probiotics!
In laboratory confirmation of the decrease of amino acids

49. In our studies: There is no decrease :
Aspargine acid, glutamic acid, ammonia;
Increases of:
-lysine, methionine, leucine, tyrosine.

50. The most frequent decrease of :
Valine, lysine, leucine, isoleucine, glutamine, tyrosine, phenylalanine, methionine, threonine (essential amino acids). It is corresponds to world studies
Increase of:
- Aspargine acid, glutamic acid, ornithine (replaced amino acids, excitatory neurotransmitters), ammonia. It is corresponds to world studies

51. It is recommended to restrict products (if glutamic acid is increased):
Curd cheese;
Eggs;
Beef, chickens, pout;
Porridges (except beech wheat, pea);
Spaghetti;
Bread (especially wheaten);
Cookies

52. It is recommended to restrict products (if asparginic acid is increased):
Eggs;
Beef;
Chickens;
Pout;
Rice, beech wheat, oatmeal;
Corn cob;
Pea

53. It is recommended to add to a diet (if there is deficiency of essential amino acids):
Curd cheese and milk products ;
Eggs;
Beef, chickens, pout;
Porridges (beech wheat, corn cob, pea);
Spaghetti;
Bread (especially wheaten);
Cookies.

54. The treatment of disorders of AA metabolism depends on disease form and the clinical picture
The most of these diseases respond to diet treatment by restriction of proteins and amino acids, involved in pathological process;
Another therapeutic tactics, which is successful in treatment of hepatorenal tyrosinemia is inhibition of biochemical reactions, which precede metabolic block;
Injection of high amounts of nicotinic acid – tryptophan cofactor (in the case of tryptophan deficiency in Hartnup disease);
Prescription of penicillamine in cystinuria prevents renal colic by development of dissoluble disulfides with cysteine

55. In the periods of acute crisis, the following is recommended:
Discontinuing of the ordinary diet;
Often introduction of drinking in a great amount.
The frequency, amount, concentration of drinking depends on children age and the main disease.
In urea cycle disorder it is necessary to increase medicines, which contribute to nitrogen release
Carnitine is usually prescribed in organic acidemias.
In disorder of branched chain-AA metabolism, their level can be decreased only because of protein formation; glucose polymers are injected for biosynthesis increase

56. In phenylketonuria – a diet with a low content of phenylalanine
Treatment diet is prescription of medicines influencing on amino acid metabolism: vitamins B, C, lipoic acid, organic acid, calcium, glycerophosphates, zinc- containing medicines
For all groups of diseases – the necessity of the individual approach to the treatment of each child

57. The deficiency in autism by data of the world literature
Metallothionein – a small protein, which is enriched with cysteine and is able to bind bivalent metals. The role of metalloprotein is the regulation of the concentration in the cell of these microelements, such as zinc and copper, and also in binding poisonous heavy metals, for example, cadmium and mercurous.

58. DAN! opinion
1. Metallothionein has to be reactivated and gradually renewed. That’s why cysteine isn’t ingested till zink and other bioelement drugs aren’t prescribed for less than the term of 3-4 months. If metalloprotein is activated too quick, deterioration can be observed, because there is upload with heavy metals in circulation pathways.
2. Cysteine, which is necessary for metallothionein, acts the most effectively in the form of glutathione (GSH). It break downs in the intestine to cysteine with minimal side effects Cysteine (GSH) in the combination with zinc and glutathione is the best way to remove excess copper and heavy metals.

59. Glutathione (2-amino-5-{[2- [(carboxymethyl)amino]- 1-(mercaptomethyl)-2- oxoethyl]amino}-oxopentanoic acid, eng. glutathione, GSH) — is tripeptide γ- glutamyl cysteinyl glycine. Glutathione contains unusual peptide connection between amino group cysteine and carboxy-group of side chain of  glutamate. The importance of glutathione in a cell is determined by its antioxidative properties. Glutathione not only defense a cell erom such toxic agents as free radicals, but also in the whole body determines redox-status of intracellular medium

60. Alternatives of the use of glutathione include N- acetylcysteine, intravenous cysteine, lipoic acid. It can be appeared more effective method of the increase of glutathione level and that’s why should be used under specialist follow-up
Side effects:some children don’t tolerate glutathione and can manifest temporary regress in behavior, especially if you began from a high dose. Nevertheless, the increase of glutathione level has significant meaning in the capacity of child’s body to detoxify.

61. Cysteine and cystine.
Can be bound with mercurous and thus release mercurous in blood again, deposited in tissues. Mercurous poisoning can be enhanced because of distribution of mercurous in other organs (possibly in brain).
Wonderful nutrition medium for yeast infections.
Cysteine level in blood in autistic children can be high .

62. N-acetyl-L- cysteine (NAC)
Can be bond with mercurous and transfer it through cell membranes.
Good nutrition medium for yeast infections
Can quickly increase intracellular level of glutathione that is very useful for regeneration of antioxidant deficiency, but it is better to use in the combination with DMSA or after the release of mercurous from blood and tissues. Use carefully for children with a high level of cysteine

63. DAN! opinion
4. Metallothionein contains many sulfur residues. Injection of additional sulfur in the form of MSM can help in regeneration of the function of metallothionein in the intestine, liver, brain. Autistic children release sulfur (in urine) 2 times higher with urine comparing with normal children, and there is only 1/5 part of normal value in blood

64. DAN! opinion
It is necessary to pay attention that autism transformation into the condition with emotional excitation can be in some cases. This can be explained by the fact that a quick increase of zinc in the intestine can lead to a quick synthesis of metallothionein, that temporary blocks zinc leading to expressed psychic excitation and hyperactivity. However, this is a sigh of regeneration!!!!

65. 12. Additionally to cysteine, metallothionein contains 13 other amino acids. Many other autistic children aren’t able to break down proteins to amino acids necessary for synthesis of metallothionein. Introduction of amino acid complexes can be an important step in treatment process. It is necessary to avoid food cooked in microwave oven, because of protein denaturation and flavonoid breakdown.

66. Right DAN recommendations :
When bioelement drugs are prescribed for regeneration of metallothionein function and a child responds (normal copper-zinc index in analyses) the following conclusion can be made: metallothionein function was degenerated. Methyl group deficiency is compensated by the use of methioneine, calcium, magnesium, vitamin B6. Calcium is very important for decrease of histamine level. Histamine hypomethylation makes normal its increase. Histamine acts as a mediator in brain.

67. From 150 children
Gender ratio was 1:3.5 (F:M), that corresponds to the world data. The main complaints were:
- psycho-speech development delay– 100%;
- the absence of visual contact and indicating gesture – 63%;
- hyperactivity, aggression– 88%;
- stereotypes – 85%;
- stool disorders (constipations,predisposition to diarrhea) – 79%;
- episyndrome – 22%
- unusual body, urine, fecal masses, sweat odor – 34%;
- frequent vomiting – 21%;
- atypical dermatitis (frequently of unknown etiology and resistant to carried out hyposensibilized therapy) – 51%.

68. By the time of manifestation:
-the first year of life – 34%;
-1-3 years– 66%.
Parents connect disease onset:
- vaccination – 31%;
- infectious diseases with antibiotic therapy – 15%;
- introduction of products with a high content of protein to the diet – 2%;
- stress – 2%;
-don’t connect with anything – 50%.

69. Features of pregnancy and delivery course:
-early toxicosis – 72%;
- anemia – 22%;
- threatened miscarriage and hormonal therapy – 47%;
- pregnancy was with the help of EF – 3%;
- genital tract infections– 49%;
-ARVI, herpetic infection, flue – 69%;
-weak delivery activity, stimulation– 66%;
-quick delivery – 18%.

70. Features of newborn period:
- prolonged conjugated jaundice – 33%;
- perinatal CNS affection– 58%;
- convulsive syndrome– 13%;
- dysbacteriosis – 45%;
- frank intertrigo – 10%.

71. Phenotype features:
- surface location of subcutaneous veins– 88%;
- paleness and dryness of skin– 90%;
- marble skin– 74%;
- hot pink palms – 87%;
- atypical dermatitis– 34%;
- skeletal changes (postural disorder,joint hypermobility, flat foot) – 76%.

72. Family history analysis:
-cardiovascular pathology– 98%;
- oncopathology – 75%;
-diabetes mellitus– 34%.
14% of children kept to free-gluten and free-casein diet during consultation and examination.

74. Proton (1Н) MR-spectroscopy
Proton (1Н) MR-spectroscopy. Decrease of the content of N-acetylaspartate (NAA) (в) in meningioma (а), comparing with a normal spectrum in opposite side (б). (Trufanov, 2013)

76. Our examination
MRS: the child is hyperactive and aggressive. Conclusion: signals of N- acetylaspartate, creatine, choline, lactate, myoinositol are in spectrums. There are signals of glutamate, glutamine and also lactate in frontotemporal areas of the right hemisphere.

77. Mother’s way Disease Official medicine New search
Mass media + experience of other mothers
Irregular examination
Confusion, uncertainty of the result
Treatment by the principle– «that helped neighbor»

78. Choosing individual diet and dietary supplements
Recommended by the metabolic specialist way
Disease
Assessment of levels of amino acids, hydrogen, lipids, microelements, vitamins, folates (qualitatively and quantitatively)
USI, NMRT, MRS
Choosing individual diet and dietary supplements

79. WE CONFIRM MENTIONED DATA BY THE EXAMPLES OF OUR OBSERVATIONS
Diagnosis
Differential diagnosis
Treatment
Effect
Biochemical
Molecular
Clinical
Diet therapy
Cofactor
Rehabilitation
Schizophrenia+С677Т MTHFR Hmzgt +
recovery
Neurofibromatosis
polymorphism -
Long remission
Tuberous sclerosis
Long remission of dispersion of tumors
Schizophrenia
Disorder of tryptophan metabolism
Full recovery
Dissecting myelitis
3 observations
Recovery, return to work
Long remission. Return to work
Autism
The child began to speak in 3 weeks after diet therapy

80. Thanks for
your attention